Pulmonary hypertension (PH) is largely incurable disease characterized by a narrowing of the small pulmonary arteries and arterioles that progressively worsens, causing substantial loss of quality of life, and significant long-term morbidity and mortality. There are 5 classes of PH recognized by the WHO, including pulmonary arterial hypertension (PAH, Class 1.1) and hypoxemia-induced PH (Class 3) for which our data implicate NOTCH3 in the vascular smooth muscle (vSMC) pathogenesis of both of these types of disease. Specifically, NOTCH3 mediates two fundamental manifestations of PH: hyperproliferation of small pulmonary artery smooth muscle cells and the excessive store-operated calcium entry (SOCE) induced by hypoxia that underlie vascular remodeling and vasoconstrictive disease phenotypes, respectively. To advance the hypothesis that NOTCH inhibition would be therapeutic, we developed highly potent and selective small molecule inhibitors of the interaction between NOTCH and its transcriptional effector RBPJ. A logical next step is to test the new NOTCH inhibitors for the ability to reverse PH symptoms using animal models in vivo and in primary cultures of vascular smooth muscle cells derived from the human small pulmonary arteries and arterioles in vitro. Recognizing that no single animal model reproduces the human condition, including gender selectivity and pathogenesis, we will use three rodent models that present the vasoconstrictive and hyperproliferative cellular phenotypes we postulate are regulated by NOTCH/RBPJ. The Specific Aims are: 1) Develop selective small molecule inhibitors of NOTCH:RBPJ to probe function in PH 2) Test whether the novel inhibitors block PH phenotypes using patient-derived small pulmonary artery smooth muscle cells (sPASMCs). 3) Evaluate whether inhibiting RBPJ reverses PH vasculopathy in animal models. In summary, this multi-PI/PD proposal builds on our complementary expertise to test the hypothesis that pharmacological inhibition of NOTCH/RBPJ can be used to successfully treat PH, which is a challenging and often incurable disease. The project is highly significant since it targets a pathway that appears to underlie pathology in multiple forms of PH. It is highly innovative since it advances the development of a new generation of selective small molecule inhibitors of NOTCH to treat PH.